CN115105600A

CN115105600A - PI3K delta/gamma medicine composition and method for treating tumor by using same

Info

Publication number: CN115105600A
Application number: CN202210121154.0A
Authority: CN
Inventors: 吕容真; 陈志宏
Original assignee: Tongrun Biomedical Shanghai Co ltd
Current assignee: Tongrun Biomedical Shanghai Co ltd
Priority date: 2021-02-10
Filing date: 2022-02-09
Publication date: 2022-09-27
Also published as: TW202241494A; CN114452403A8; EP4292596A1; CN114452403A; US20240139198A1; WO2022171121A1; CN117222413A

Abstract

The present application relates to a pharmaceutical combination comprising a phosphoinositide 3-kinase (PI3K) inhibitor and a second therapeutic agent, wherein the second therapeutic agent is an inhibitor of an immune checkpoint, a TGF inhibitor, a bifunctional immune checkpoint/TGF inhibitor or a combination thereof. The application also provides methods of treatment comprising administering the combination, and the use of the pharmaceutical combination for treating a tumor.

Description

PI3K delta/gamma medicine composition and method for treating tumor by using same

Technical Field

The application relates to the field of biomedicine, in particular to a method and a combination for treating tumors.

Background

The phosphoinositide 3-kinase (PI3K) signal transduction pathway is one of the most highly mutated systems in human cancers. PI3K is a member of a unique and conserved family of intracellular lipid kinases that phosphorylate the 3' -OH group on phosphatidylinositol or phosphoinositides. The PI3K family includes 15 kinases with different substrate specificities, expression patterns and regulatory patterns. Class I PI3K (p110 α, p110 β, p110 δ and p110 γ) are typically activated by tyrosine kinases or G-protein coupled receptors to produce (3,4,5) -phosphotidylinositol triphosphate (PIP3), which engages downstream effectors such as those in the AKT/PDK1 pathway, mTOR, Tec family kinases and Rho family gtpases. Class II and class III PI3K play a key role in intracellular trafficking by synthesizing phosphatidylinositol 3-diphosphate (PI (3) P) and phosphatidylinositol (3,4) -diphosphate (PI (3,4) P2). PI3K is a protein kinase that controls cell growth (mTORC1) or monitors genomic integrity (ATM, ATR, DNA-PK, and hSmg-1).

There are four mammalian subtypes of class I PI 3K: PI3K- α, β, δ (class Ia PI3K) and PI3K- γ (class Ib PI 3K). These enzymes catalyze the production of PIP3, resulting in the activation of downstream effector pathways important to cell survival, differentiation, and function. PI 3K-alpha and PI 3K-beta are widely expressed and are important mediators of signal transduction from cell surface receptors. PI 3K-alpha is the most common subtype of mutation in cancer and plays a role in insulin signaling and glucose homeostasis (Knight et al Cell (2006)125(4): 733-47; Vanhasebreeck et al Current Topic Microbiol. Immunol. (2010)347: 1-19). PI3K- β is activated in cancers with deletions of phosphatase and tensin homolog (PTEN). Both subtypes are cancer targets for small molecule therapies under development.

PI3K- δ and- γ are preferentially expressed in leukocytes and are important in leukocyte function. These subtypes also contribute to the development and maintenance of hematological malignancies (Vanhaaesebroeck et al Current Topic Microbiol. Immunol. (2010)347: 1-19; Clayton et al J Exp Med. (2002)196(6) 753-63; Long-Leung Cell Signal. (2011)23(4) 603-8; Okkenhaug et al Science (2002)297(5583) 1031-34). PI3K- δ is activated by a cellular receptor (e.g., receptor tyrosine kinase) either by interaction with the Sarc homology 2(SH2) domain of the PI3K regulatory subunit (p85) or by direct interaction with the RAS.

Transforming growth factor beta (TGF β) is a potent cytokine with a significant impact on the immune system. The primary function of TGF β in the immune system is to maintain tolerance and initial immune response to foreign pathogens. Three isoforms of TGF β have been identified in mammals, namely TGF β 1, TGF β 2 and TGF β 3, with TGF β 1 being the predominant isoform. TGF β is secreted in a latent form, and only a small fraction of all TGF β secreted is activated under physiological conditions. The biological effects of TGF β are largely achieved by binding of TGF β to the receptors ALK5 and TGF β receptor II (TGF β R2). In particular, active TGF β dimers bind to the tetrameric ALK5 and TGF β R2 complexes to initiate signal transduction. ALK5 is not required for initial binding of TGF β, but is required for signaling.

Programmed death receptor 1(PD-1) is a member of the CD28 superfamily. PD-1 is expressed in activated T cells, B cells and myeloid lineage cells and has two ligands, programmed death ligand-1 (PD-L1) and PD-L2. PD-L1 interacts with the receptor PD-1 on T cells and plays an important role in the negative regulation of the immune response. The expression of PD-L1 protein can be detected in a plurality of human tumor tissues, the microenvironment of the tumor part can induce the expression of PD-L1 on tumor cells, and the expressed PD-L1 is beneficial to the generation and growth of tumors and induces the apoptosis of anti-tumor T cells. The PD-1/PD-L1 pathway inhibitor can block the combination of PD-1 and PD-L1, block negative regulation signals, restore the activity of T cells, and enhance immune response

Transforming growth factor-beta (TGF-beta) belongs to the TGF-beta superfamily that regulates cell growth and differentiation. TGF-. beta.signals through a heterotetrameric receptor complex consisting of two type I and two type II transmembrane serine/threonine kinase receptors.

TGF β can affect a number of cellular functions such as cell proliferation, differentiation, cell-cell and cell-matrix adhesion, cell motility, and activation of lymphocytes. (for a review of the role of TGF-beta in modulating the immune response, see Li et al (2006) Annu. Rev. Immunol.24: 99-146.) furthermore, it is believed that TGF-beta can induce or mediate the progression of a number of diseases, such as osteoporosis, hypertension, atherosclerosis, cirrhosis of the liver, fibrotic diseases of the kidney, liver and lung, and tumor progression. TGF β can enhance end organ damage caused by chronic inflammation in animal models of certain diseases such as diabetic nephropathy, glomerulonephritis, cyclosporin-mediated kidney injury and Systemic Lupus Erythematosus (SLE), and TGF β antagonists can effectively alleviate such damage (Border et al (1990) Nature 346: 371- > 374; Border et al (1992) Nature 360: 361- > 364; Isaka et al (1999) Kidney Int.55: 465- > 475; Sharma et al (1996) Diabetes 45: 522; Xin et al (2004) Transplantation 15: 1433; Benigi et al (2003) J.am. Soc. Nephrol.14: 1816). In cancer, TGF β may have a direct inhibitory effect on malignant cells and may increase the production or activity of a range of tumor growth factors and angiogenic factors.

In the treatment of tumors, chemotherapy has long been recognized as highly toxic and may cause adverse effects on drug resistant cancer cells. Even with therapies targeting overexpressed or activated proteins associated with tumor survival and growth, cancer cells can mutate to reduce or escape the dependence on the pathway targeted by the targeted therapy and continue to survive using other pathways. The tumor immunotherapy mainly improves the immunogenicity of tumor cells and the sensitivity to killing of effector cells through an immunological principle and a method, stimulates and enhances the anti-tumor immune response of an organism, and applies immune cells and effector molecules to be infused into a host body to cooperate with an immune system of the organism to kill tumors and inhibit the growth of the tumors. The toxicity and the resistance of the tumor treatment drug are reduced, and the toxic and side effects are reduced, so that the problem to be solved urgently in tumor treatment is still solved.

Disclosure of Invention

It is an object of the present application to provide combinations and methods comprising a PI3K inhibitor with a selected second therapeutic agent. In certain embodiments, the combination of a PI3K inhibitor and a second therapeutic agent selected from one or more of the following has been found to have a synergistic effect in treating tumors (e.g., in reducing cancer cell growth or viability or both): an inhibitor of an immune checkpoint, a TGF β inhibitor, or a combination thereof. The combination of the PI3K inhibitor and the selected second therapeutic agent can allow the PI3K inhibitor, the second therapeutic agent, or both to be administered at a lower dose than would be required to achieve the same therapeutic effect as a monotherapy dose. In some embodiments, the combination may allow the PI3K inhibitor, the second therapeutic agent, or both to be administered less frequently than when the PI3K inhibitor or the second therapeutic agent is administered as monotherapy. Such combinations may provide beneficial effects, for example in reducing, preventing, delaying and/or reducing the occurrence of one or more of the following: side effects, toxicity or resistance that would otherwise be associated with administration of higher doses of the agent.

In one aspect, the present application provides a pharmaceutical combination comprising a phosphoinositide 3-kinase (PI3K) inhibitor and a second therapeutic agent, wherein the second therapeutic agent is a bifunctional immune checkpoint/TGF β inhibitor or a combination thereof.

In certain embodiments, the bifunctional immune checkpoint/TGF inhibitor is selected from the group consisting of a PD-L1/TGF dual inhibitor and a PI3K inhibitor in combination; or a combination of a dual PD-1/TGF-beta inhibitor and a PI3K inhibitor.

In certain embodiments, wherein the dual PD-L1/TGF β inhibitor is a fusion protein.

In certain embodiments, wherein the PD-L1// TGF β dual inhibitor comprises a PD-L1 targeting moiety and a TGF β receptor domain comprising transforming growth factor β receptor II (TGF β RII) or a functionally active fragment thereof.

In certain embodiments, wherein the PD-L1// TGF dual inhibitor comprises a polypeptide, wherein the polypeptide comprises at least: (i) the heavy chain variable region of an anti-PD-L1 antibody; and (ii) TGF β RII or a functionally active fragment thereof.

In certain embodiments, wherein the anti-PD-L1 antibody heavy chain variable region comprises HCDR1, HCDR2, HCDR 3; wherein the HCDR1 comprises an amino acid sequence having at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO. 1, the HCDR2 comprises an amino acid sequence having at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO. 2, and the HCDR3 comprises an amino acid sequence having at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO. 3; or

Wherein the HCDR1, HCDR2, HCDR3 have at least about 80% sequence identity to HCDR1, HCDR2, HCDR3, respectively, of the following molecules: atezolizumab, Avelumab, BMS-936559, MPDL3280A (RG7446) or durvalumab (MEDI-4736).

In certain embodiments, wherein the anti-PD-L1 antibody heavy chain variable region comprises HCDR1, HCDR2, HCDR3, said HCDR1 comprises the amino acid sequence set forth in SEQ ID No. 1 or an amino acid sequence obtained by an amino acid addition, deletion or substitution reaction having NO more than 2 amino acid differences from the amino acid sequence set forth in SEQ ID No. 1; the HCDR2 comprises an amino acid sequence shown in SEQ ID NO. 2 or an amino acid sequence which is obtained by amino acid addition, elimination or substitution reaction and has NO more than 2 amino acid differences with the amino acid sequence shown in SEQ ID NO. 2; the HCDR3 comprises an amino acid sequence shown in SEQ ID NO. 3 or an amino acid sequence which is obtained by amino acid addition, elimination or substitution reaction and has NO more than 2 amino acid differences with the amino acid sequence shown in SEQ ID NO. 3.

In certain embodiments, wherein the anti-PD-L1 antibody heavy chain variable region comprises an amino acid sequence selected from the group consisting of seq id nos: (a) 4, an amino acid sequence shown as SEQ ID NO; (b) an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 4; (c) an amino acid sequence having 1 or more differences from the amino acid sequence shown in SEQ ID NO. 4, which is obtained by addition, elimination or substitution reaction; and (d) an amino acid sequence having at least about 80% sequence identity to the heavy chain variable region of: atezolizumab, Avelumab, BMS-936559, MPDL3280A or durvalumab.

In certain embodiments, wherein the TGF β RII or functionally active fragment thereof comprises:

(a) an amino acid sequence shown as SEQ ID NO. 6;

(b) an amino acid sequence having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO 6; or

(c) Has an amino acid sequence with one or more amino acids added, deleted and/or substituted compared with the amino acid sequence shown in SEQ ID NO. 6.

In certain embodiments, wherein said polypeptide further comprises a linker linking the C-terminus of the heavy chain variable region of said anti-PD-L1 antibody or antigen-binding fragment thereof to the N-terminus of said TGF β RII or functionally active fragment thereof.

In certain embodiments, the polypeptide further comprises CH2, CH3 domain, the polypeptide comprising, in order from N-terminus to C-terminus, the heavy chain variable region (VH) of the anti-PD-L1 antibody, CH2, CH3 domain, and TGF β RII or a functionally active fragment thereof, the C-terminus of the CH3 domain being linked to the N-terminus of the TGF β RII or a functionally active fragment thereof by a linker.

In certain embodiments, preferably the CH2, CH3 domains are derived from IgG.

In certain embodiments, wherein the linker is a peptide linker.

In certain embodiments, wherein the amino acid sequence of the peptide linker is (G) ₄ S) _x Wherein x is any integer from 3 to 6.

In certain embodiments, wherein the peptide linker comprises an amino acid sequence selected from the group consisting of seq id nos: (a) the amino acid sequence shown as SEQ ID NO. 5; (b) an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 5; and (c) an amino acid sequence having 1 or more difference from the amino acid sequence shown in SEQ ID NO. 5, which is obtained by addition, elimination or substitution reaction.

In certain embodiments, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of seq id no: (a) the amino acid sequence shown as SEQ ID NO. 7; (b) an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 7; (c) an amino acid sequence having 1 or more differences from the amino acid sequence shown in SEQ ID NO. 7, which is obtained by addition, deletion or substitution reaction.

In certain embodiments, wherein the PI3K inhibitor is a PI3K delta/gamma dual inhibitor.

In certain embodiments, wherein the PI3K inhibitor comprises 2- (1- (9H-purin-6-ylamino) propyl) -3- (3-fluorophenyl) -4H-chromen-4-one, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof.

In certain embodiments, wherein the PI3K inhibitor comprises (S) -2- (1- (9H-purin-6-ylamino) propyl) -3- (3-fluorophenyl) -4H-chromen-4-one, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof.

In certain embodiments, the second therapeutic agent and the PI3K inhibitor are not intermixed or are independently present in separate containers in the pharmaceutical combination.

In another aspect, the present application provides the use of the aforementioned pharmaceutical combination or the aforementioned pharmaceutical composition for the manufacture of a medicament for the treatment and/or prevention of a tumor.

In certain embodiments, wherein the tumor comprises a solid tumor and a non-solid tumor.

In certain embodiments, wherein the tumor comprises a tumor with aberrant PI3K expression.

In certain embodiments, wherein the tumor comprises a tumor with aberrant PD-L1 expression.

In certain embodiments, wherein the tumor comprises a tumor with aberrant TGF β expression.

In certain embodiments, the tumor comprises a tumor of the digestive tract, melanoma or lymphoma, or breast cancer.

In certain embodiments, wherein the tumor has a delayed resistance to the PI3K inhibitor.

In certain embodiments, wherein remission of the tumor in the subject is prolonged.

In certain embodiments, wherein the subject experiences complete remission of the tumor.

In certain embodiments, wherein the level of Minimal Residual Disease (MRD) is decreased.

For example, the use of an inhibitor of the aforementioned PI3K and an inhibitor of the aforementioned immune checkpoint in the manufacture of a medicament for the treatment and/or prevention of a tumor. For example, the use of an aforementioned PI3K inhibitor and an aforementioned TGF β inhibitor in the manufacture of a medicament for the treatment and/or prevention of a tumour. For example, the use of an aforementioned PI3K inhibitor, an aforementioned inhibitor of an immune checkpoint and an aforementioned TGF β inhibitor in the manufacture of a medicament for the treatment and/or prevention of a tumour.

In another aspect, the present application provides a pharmaceutical combination of the foregoing or a pharmaceutical composition of the foregoing for use in the treatment and/or prevention of a tumor.

In another aspect, the present application provides a method of inhibiting tumor growth, comprising contacting a tumor with the aforementioned pharmaceutical combination or the aforementioned pharmaceutical composition.

In certain embodiments, the contacting is in an in vitro or ex vivo environment.

In certain embodiments, the method comprises contacting the tumor with the aforementioned inhibitor of PI3K and the aforementioned inhibitor of an immune checkpoint. In certain embodiments, the method comprises contacting the tumor with the aforementioned PI3K inhibitor and the aforementioned TGF β inhibitor. In certain embodiments, the method comprises contacting the tumor with the aforementioned PI3K inhibitor, the aforementioned inhibitor of an immune checkpoint, and the aforementioned TGF β inhibitor.

In another aspect, the present application provides a method of inhibiting tumor growth, comprising contacting a tumor with the aforementioned PI3K inhibitor and the aforementioned dual PD-L1/TGF β inhibitor.

In another aspect, the present application provides a kit comprising: (1) a first container, and the aforementioned PI3K inhibitor located in the first container; (2) a second container, and the aforementioned dual PD-L1/TGF β inhibitor located in the second container.

In another aspect, the present application provides a kit that can include: (1) a first container, and the aforementioned PI3K inhibitor located in the first container; (2) a second container, and an inhibitor of the aforementioned immune checkpoint or an inhibitor of the aforementioned TGF β located in said second container.

In certain embodiments, the kit further comprises (3) a third container, wherein the agent in the third container is different from the agent in the second container, and an inhibitor of an immune checkpoint as described above or a TGF β inhibitor as described above is located in the third container.

In certain embodiments, the drug in the kit is in an oral dosage form or an injectable dosage form.

In certain embodiments, the kit further comprises instructions.

Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.

Drawings

Specific features of the invention to which this application relates are set forth in the following claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The drawings are briefly described as follows:

FIGS. 1A-1F show the results of combinations of CN401(Tenalisib) (150mg/kg) and CN202(WBP1126) (5mg/kg) in an A20 mouse B-cell lymphoma animal model.

FIGS. 2A-2F show the results of combinations of CN401(Tenalisib) (150mg/kg) and CN202(WBP1126) (15mg/kg) in an A20 mouse B-cell lymphoma animal model.

FIGS. 3A-3B show the results of combinations of CN401(Tenalisib) (150mg/kg) and CN202(WBP1126) (5mg/kg)/(15mg/kg) in an A20 mouse B-cell lymphoma animal model.

FIGS. 4A-4F show the results of CN202(WBP1126) (5mg/kg) in combination with CN401(Tenalisib) (150mg/kg) in the A20 mouse B cell lymphoma animal model.

FIGS. 5A-5F show the results of combinations of CN202(WBP1126) (5mg/kg) with Alpelisib (50mg/kg) in an A20 mouse B-cell lymphoma animal model.

FIGS. 6A-6F show the results of the combination of CN401(Tenalisib) (150mg/kg) with anti-PD-L1 antibody (Atezolizumab) (5mg/kg) in an A20 mouse B-cell lymphoma animal model.

FIGS. 7A-7B show the results of CN401(Tenalisib) (150mg/kg), CN202(WBP1126) (15mg/kg) in combination with albumin paclitaxel (nab-pac) (10mg/kg) in a mouse EMT-60 (mouse breast cancer cell) tumor animal model.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.

Definition of terms

In the present application, a "phosphoinositide 3-kinase (PI3K) inhibitor" or "PI 3K inhibitor" refers generally to any inhibitor of PI 3K. PI3K is a member of a unique and conserved family of intracellular lipid kinases that phosphorylate the 3' -OH group on phosphatidylinositol or phosphoinositides. The PI3K family includes kinases with diverse substrate specificity, expression patterns, and regulatory patterns (see, e.g., Katso et al, 2001, Annu. Rev. Cell Dev. biol.17, 615-675; Foster, F.M. et al, 2003, J Cell Sci 116, 3037-3040). Class I PI3K (e.g., P110 α, P110 β, P110 γ, and P110 δ are typically activated by tyrosine kinases or G-protein coupled receptors to produce PIP3, PIP3 binds downstream mediators such as those in the Akt/PDK1 pathway, mTOR, Tec family kinases, and Rho family gtpase class II PI3K (e.g., PI3K-C2 α, PI3K-C2 β, PI3K-C2 γ) and class III PI3K (e.g., Vps34) play a key role in intracellular trafficking by synthesizing PI (3) P and PI (3,4) P2 specific exemplary PI3K inhibitors, e.g., PI3K inhibitors inhibit PI3K- α, PI3K- β, PI3K- γ, and PI3K- δ subtypes, or combinations thereof.

For example, the PI3K inhibitor can be the PI3K delta/gamma dual inhibitor Tenalisib, 2- (1- (9H-purin-6-ylamino) propyl) -3- (3-fluorophenyl) -4H-chromen-4-one, having the formula:

for example, the PI3K inhibitor may be the PI3K inhibitor Alpelisib, (2S) -1-N- [ 4-methyl-5- [2- (1,1, 1-trifluoro-2-methylpropan-2-yl) pyridin-4-yl ] -1, 3-thiazol-2-yl ] pyrrolidine-1, 2-dicarboxamide, having the formula:

in some embodiments, a PI3K inhibitor generally refers to a compound that inhibits one or more PI3K subtypes with an IC50 of less than about 1000nM, less than about 900nM, less than about 800nM, less than about 700nM, less than about 600nM, less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, less than about 100nM, less than about 75nM, less than about 50nM, less than about 25nM, less than about 20nM, less than about 15nM, less than about 10nM, less than about 5nM, or less than about 1 nM.

In the present application, the term "immune checkpoint" generally refers to a set of molecules on the cell surface of CD4 cells and CD8T cells. These molecules can effectively act as "brakes" that down-regulate or inhibit the anti-tumor immune response. Immune checkpoint molecules include, but are not limited to, programmed cell death 1(PD-1), programmed cell death ligand 1(PD-L1), programmed cell death ligand 2(PD-L2), lymphocyte activation gene-3 (LAG-3; also known as CD223), galectin-3, B and T Lymphocyte Attenuator (BTLA), T-cell membrane protein 3(TIM3), galectin-9 (GAL9), B7-H1, B7-H3, B7-H4, T-cell immunoreceptor with Ig and ITIM domains (TIGIIT/Vstm 3/WUCAM/VSIG9), T-cell activated V-domain Ig inhibitor (VISTA), glucocorticoid-induced tumor necrosis factor receptor-related (GITR) protein, Herpes Virus Entry Mediator (HVEM), 40, OX, CD27, CD28, CD80, CD86, CD137, CGEN-15001T, CGEN-15022, CGEN-15027, CGEN-15049, CGEN-15052, and CGEN-15092.

In the present application, the term "inhibitor of an immune checkpoint" generally refers to a molecule that inhibits, reduces or interferes with the activity of an inhibitory checkpoint molecule. Without being bound by a particular theory, inhibitory checkpoint molecules down-regulate immune responses (e.g., T-cell activation) by delivering a negative signal to T cells upon their binding by a ligand or anti-receptor. In certain embodiments, checkpoint inhibitors for use with the methods and compositions provided herein can directly inhibit the activity of an inhibitory checkpoint molecule or reduce the expression of an inhibitory checkpoint molecule or interfere with the interaction of an inhibitory checkpoint molecule and a binding partner (e.g., a ligand).

In the present application, an "inhibitor of an immune checkpoint" includes, but is not limited to, a protein, polypeptide, peptide, antisense oligonucleotide, antibody fragment, or RNA molecule (e.g., an inhibitory RNA molecule that targets the expression of an inhibitory checkpoint molecule). The inhibition can be at the DNA, RNA or protein level. For example, inhibitory nucleic acids (e.g., dsRNA, siRNA or shRNA) can be used to inhibit expression of inhibitory molecules. In other embodiments, the inhibitor of an inhibitory signal is a polypeptide that binds to an immune checkpoint, e.g., a soluble ligand (e.g., PD-1-Ig), an antibody or antigen-binding fragment thereof; for example, an antibody or fragment thereof that binds to PD-1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and/or TGFR β, or a combination thereof.

In the present application, the term "TGF β RII" or "TGF β receptor II" generally refers to a polypeptide having a wild-type human type 2 TGF β receptor isoform a sequence (e.g., the amino acid sequence of NCBI reference sequence (Ref Seq) accession No. NP _ 001020018), or a wild-type human type 2 TGF β receptor isoform B sequence (e.g., the amino acid sequence of NCBI Ref Seq accession No. NP _ 003233) or a polypeptide having substantially the same sequence as their amino acid sequence. The tgfbetarii may retain at least 0.1%, 0.5%, 1%, 5%, 10%, 25%, 35%, 50%, 75%, 90%, 95% or 99% of the wild-type sequence tgfbeta binding activity.

In the present application, the term "TGF β RII fragment capable of binding TGF β" generally refers to any portion of NCBI Ref Seq accession No. NP _001020018 or NCBI Ref Seq accession No. NP _003233, or sequences substantially identical thereto, fragments at least 20 (e.g., at least 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 175, or 200) amino acids in length and retaining at least a portion of the TGF β binding activity (e.g., at least 0.1%, 0.5%, 1%, 5%, 10%, 25%, 35%, 50%, 75%, 90%, 95%, or 99%) of a wild-type receptor or wild-type fragment thereof. Typically, these fragments are soluble fragments. One of the exemplary fragments is the extracellular domain of TGF-beta RII having the sequence of SEQ ID NO 6.

In the present application, "substantially identical" generally means that the polypeptide exhibits at least 50%, preferably 60%, 70%, 75% or 80%, more preferably 85%, 90% or 95%, and most preferably 99% amino acid sequence identity (identity) with the reference amino acid sequence. The length of the comparison sequences is generally at least 10 amino acids, preferably at least 15 contiguous amino acids, more preferably at least 20, 25, 50, 75, 90, 100, 150, 200, 250, 300 or 350 contiguous amino acids, and most preferably the full-length amino acid sequence. For example, suitable algorithms for determining percent sequence identity and percent sequence similarity can be the BLAST and BLAST 2.0 algorithms, see Altschul et al (1977) nucleic acids Res.25: 3389 and Altschul et al (1990) J.mol.biol.215: 403.

in the present application, the term "TGF β inhibitor" generally refers to a substance that inhibits TGF β signaling molecule production and signal transduction, and the inhibitor may be a small molecule compound or a large molecule compound (e.g., an antibody). Exemplary small molecule TGF β inhibitors include SB525334, SD-208, SB431542, LY2109761, LY2157299 (Galunesertib), GW788388, Repsox, SIS3, LDN-193189, EW-7197, LY364947 and the like.

In the present application, the term "antibody" generally refers to an immunoglobulin that is reactive with a specified protein or peptide or fragment thereof. The antibody can be an antibody from any class, including but not limited to IgG, IgA, IgM, IgD, and IgE, and antibodies from any subclass (e.g., IgG1, IgG2, IgG3, and IgG 4). The antibody may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG 4. The antibody may also have a light chain selected from, for example, kappa (. kappa.) or lambda (. lamda.). The antibodies of the present application may be derived from any species.

In the present application, the term "antigen-binding fragment" generally refers to a portion of an antibody molecule that comprises amino acids responsible for specific binding between an antibody and an antigen. The portion of the antigen specifically recognized and bound by the antibody is referred to as an "epitope" as described above. As described above, the antigen binding domain may typically comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not necessarily include both. Fd fragments, for example, have two VH regions and typically retain some of the antigen binding function of the intact antigen binding domain. Examples of antigen-binding fragments of antibodies include (1) Fab fragments, monovalent fragments having VL, VH, constant light Chain (CL) and CH1 domains; (2) a F (ab') 2 fragment, a bivalent fragment having two Fab fragments linked by a disulfide bridge of the hinge region; (3) an Fd fragment having two VH and CH1 domains; (4) fv fragments having VL and VH domains of a Single arm of an antibody, (5) dAb fragments (Ward et al, "Binding Activities of a repertile of Single Immunoglobulin Variable domain derived From Escherichia coli," Nature 341:544-546(1989), which is incorporated herein by reference in its entirety), having VH domains; (6) an isolated Complementarity Determining Region (CDR); (7) single chain fv (scFv), e.g.from a scFv-library. Although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by synthetic linkers that allow them to be prepared as a Single Protein chain in which the VL and VH regions pair to form monovalent molecules (known as Single chain Fv (scFv)) (see, e.g., Huston et al, "Protein Engineering of antibody binding Sites: Recovery of Specific Activity in an Anti-Di-goxin Single-chain-ChainFv antibody Produced in Escherichia coli," Proc. Natl.Acad.Sci.USA 85: 5879-; (8) "VHH" relates to The variable antigen binding domain of heavy chain antibodies from camelidae (camel, dromedary, llama, alpaca, etc.) (see Nguyen v.k. et al 2000, The EMBO Journal, 19, 921-930;

muydermans S.2001, J Biotechnol., 74, 277-. VHH may also be referred to as Nanobody (Nb).

In the present application, the term "variable region" or "variable domain" generally refers to the domain of an antibody heavy or light chain that is involved in the binding of an antibody to an antigen. In the present application, the term "variable" generally means that certain portions of the sequence of the variable domains of an antibody vary strongly, resulting in the binding and specificity of each particular antibody for its particular antigen. The variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the light and heavy chain variable regions, called Complementarity Determining Regions (CDRs) or hypervariable regions (HVRs), LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, respectively. The more highly conserved portions of the variable domains are called the Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4), largely in a beta-sheet configuration, connected by three CDR structural loop regions. The CDRs in each chain are held in close proximity by the FR regions and form, together with the CDRs from the other chain, the antigen binding site of the antibody.

In the art, variable regions of antibodies or CDRs of partitioned antibodies can be encoded by a variety of methods, such as Kabat Numbering scheme and Definition rules based on sequence variability (see Kabat et al, immunological protein sequences, fifth edition, national institutes of health, Besserda, Md. (1991)), Chothia Numbering scheme and Definition rules based on the position of structural loop regions (see, A1-Lazikani et al, Jmol Biol 273:927-48,1997), IMGT Numbering scheme and Definition rules based on alignment of amino acid sequences of germline V genes by efranc et al, as well as Honneger's Numbering scheme (AHo's), Martin Numbering scheme, Gelfand Numbering scheme, etc., see Mathieu Donglinger et al, expressed Signaling and interactions of Antibody Binding-Binding, Surface, and detail, 2018.

In the present application, "peptide linker" generally refers to an amino acid sequence that: the amino acid sequences of the different domains of the dual PD-L1/TGF β inhibitor of the present application are linked to one another by said amino acid sequences. An essential technical feature of such a peptide linker is that the peptide linker does not comprise any polymerization activity. Preferred amino acid residues for the peptide linker include Gly, Ser and Thr, characterized by a length of 5 to 25 amino acid residues. Suitable peptide linkers include those described in U.S. Pat. Nos. 4,751,180 and 4,935,233 or WO 88/09344. A preferred embodiment of the peptide linker is characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e. Gly ₄ Ser, or polymers thereof, i.e. (Gly) ₄ Ser) x, wherein x is an integer of 1 or greater. The characteristics of the peptide linkers, including lack of promoting secondary structure, are known in the art and are described, for example, in Dall' Acqua et al (Biochem. (1998)37,9266-9273), Cheadle et al (MolImmunol (1992)29,21-30), and Raag and Whitlow (FASEB (1995)9(1), 73-80). Peptide linkers that do not also promote any secondary structure are preferred. As described in the examples, the linking of the domains to each other is provided, for example, by genetic engineering. For making fusion and operably linking bispecific single chain constructs and in mammalsMethods for expressing them in animal cells or bacteria are well known in the art (e.g., WO99/54440 or Sambrook et al, Molecular Cloning: Arabidopsis Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

In the present application, the terms "antagonist" and "inhibitor" are used interchangeably and generally refer to a compound or agent that is capable of reducing or inhibiting a biological function of a target protein or polypeptide, such as reducing or inhibiting the activity or expression of the target protein or polypeptide. The inhibitor need not completely eliminate the biological function of the target protein or polypeptide, and in some embodiments, reduces activity by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%. While some antagonists of the present application specifically interact with (e.g., bind to) a target, compounds that inhibit the biological activity of a target protein or polypeptide by interacting with other members of a signal transduction pathway that includes the target protein or polypeptide are also specifically included in this definition. Non-limiting examples of biological activities that are inhibited by antagonists include those activities associated with the development, growth or spread of tumors or unwanted immune responses manifested in autoimmune diseases.

In the present application, the term "effective amount" or "therapeutically effective amount" generally refers to an amount of a compound or pharmaceutical composition described herein sufficient to achieve the intended use described below, including but not limited to disease treatment. The therapeutically effective amount may vary according to: intended applications (in vivo or in vitro); or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition; modes of administration, and the like, which can be readily determined by one of ordinary skill in the art. The term also applies to doses that will induce a specific response in the target cells, such as platelet adhesion and/or cell migration. The specific dosage will vary, for example, according to the following: the particular compound selected, the dosing regimen followed, whether or not to be administered in combination with other agents, the time of administration, the tissue to be administered, and the physical delivery system in which it is delivered.

In this application, the term "in vivo" generally refers to an event that occurs in a subject.

In the present application, the term "in vitro" generally refers to an event that occurs outside of the body of a subject. For example, an in vitro assay includes any assay performed outside of a subject. In vitro assays include cell-based assays, wherein live or dead cells are employed. In vitro assays also include cell-free assays, wherein intact cells are not employed.

In the present application, "combination therapy" or "combination" generally refers to the use of more than one compound or agent to treat a particular disorder or condition. For example, the PI3K inhibitor may be administered in combination with at least one additional therapeutic agent. "combination" is not intended to imply that other therapies and PI3K inhibitors must be administered simultaneously and/or formulated for delivery together, but these methods of delivery are within the scope of the present application. The PI3K inhibitor can be administered concurrently with, before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks before) or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks after) one or more additional agents. Generally, each therapeutic agent will be administered at a dose and/or on a schedule determined for that particular agent. The other therapeutic agents may be administered in a single composition with the PI3K inhibitor provided herein or separately in different compositions. Higher combinations are also contemplated herein, such as triple therapy of PI3K inhibitor + PD-L1 inhibitor + TGF β inhibitor.

Combination therapy the term "administering" generally refers to introducing the drug combination into the body of a subject by any route of introduction or delivery. Any method known to those skilled in the art for contacting a cell, organ or tissue with the drug combination may be employed. Including but not limited to intra-arterial, intranasal, intra-abdominal, intravenous, intramuscular, subcutaneous, transdermal, or oral. A daily dose may be divided into one, two or more doses of suitable form to be administered at one, two or more times during a certain period of time.

In certain embodiments, the term "administering" includes administering two or more agents to a subject such that the agents and/or their metabolites are present in the subject at the same or substantially the same time. In certain embodiments, co-administration of a PI3K inhibitor with an additional anti-cancer agent (both components are hereinafter referred to as "both active agents") refers to any administration, separate or together administration of the two active agents, wherein the two active agents are administered as part of a suitable dosage regimen intended to achieve the benefits of the combination therapy. Thus, the two active agents may be administered as part of the same pharmaceutical composition or in separate pharmaceutical compositions. The additional agents may be administered prior to, concurrently with, or subsequent to administration of the PI3K inhibitor, or in some combination thereof. When a PI3K inhibitor is administered to a patient, e.g., at repeated intervals during a standard course of therapy, the additional agent can be administered before, concurrently with, or after each administration of the PI3K inhibitor, or in some combination thereof, or at different intervals relative to PI3K inhibitor treatment, or in a single dose before a course of therapy with the PI3K inhibitor, at any time during a course of therapy with the PI3K inhibitor, or after a course of therapy with the PI3K inhibitor. In certain embodiments, the first agent can be administered prior to (e.g., prior to 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks), substantially simultaneously with or after (e.g., after 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) the administration of the second therapeutic agent.

In the present application, "monotherapy" refers to the use of an agent alone (also referred to herein as alone (alone)), e.g., as a single compound or agent, e.g., in the absence of a second active ingredient, for the treatment of the same indication, e.g., cancer. For example, in this context, the term monotherapy encompasses the use of a PI3K inhibitor or a second agent alone to treat cancer.

In the present application, the term "synergistic effect" or "synergistic" includes a greater additive effect of a combination of two or more agents as compared to the individual effects of the two or more agents. In certain embodiments, synergistic or synergistic refers to the beneficial effect of using two or more agents in combination, e.g., in a pharmaceutical composition or in a method of treatment. In certain embodiments, one or more beneficial effects are achieved by using a PI3K inhibitor in combination with a second therapeutic agent (e.g., one or more second therapeutic agents) described herein.

In some embodiments, synergistic effect refers to a lower dose of one or both agents required to achieve an effect. For example, a combination may provide a selected effect, e.g., a therapeutic effect, when at least one agent is administered at a lower dose than the dose of the agent required to achieve the same therapeutic effect when the agent is administered as a monotherapy. In certain embodiments, the combination of a PI3K inhibitor (e.g., a PI3K inhibitor) and a second agent (as described herein) allows the PI3K inhibitor to be administered at a lower dose than is required to achieve the same therapeutic effect when the PI3K inhibitor is administered as a monotherapy.

In some embodiments, the synergistic effect reduces, prevents, delays or reduces the occurrence or likelihood of the occurrence of one or more side effects, toxicity, resistance, or the like, that would otherwise be associated with the administration of at least one of the agents.

In some embodiments, the synergistic effect reduces resistance to at least one of the agents (e.g., a measure of resistance decreases or the likelihood of developing resistance decreases) or delays development of resistance to at least one of the agents.

In some embodiments, the synergistic effect is a reduction in Minimal Residual Disease (MRD). In certain embodiments, the combination of a PI3K inhibitor (e.g., a PI3K inhibitor described herein) and a second agent (e.g., a second agent described herein) is effective to reduce MRD in the subject, e.g., below a level previously measured in the subject (e.g., a level measured prior to administration of the combination). In certain embodiments, the combination of the PI3K inhibitor and the second agent is effective to reduce MRD in the subject below levels observed during or after administration of monotherapy treatments, e.g., monotherapy comprising either the PI3K inhibitor or the second agent. In certain embodiments, the MRD is reduced below the levels observed during monotherapy treatment with a PI3K inhibitor. In certain embodiments, the MRD is reduced below the level observed during treatment with a monotherapy comprising a second agent. In certain embodiments, the combination is effective to reduce the level of MRD below a preselected cutoff value (e.g., 1 malignant cell out of 100 normal cells, 1 malignant cell out of 1000 normal cells, or 1 malignant cell out of 10,000 normal cells, or 1 malignant cell out of 100,000 normal cells). In certain embodiments, the preselected cutoff value is 1 malignant cell out of 1000 normal cells. In certain embodiments, the preselected cutoff value is 1 malignant cell out of 100,000 normal cells.

In some embodiments, synergistic means that the combination of a PI3K inhibitor (e.g., a PI3K inhibitor or a pharmaceutically acceptable form thereof) and a second therapeutic agent (e.g., one or more additional therapeutic agents or a pharmaceutically acceptable form thereof) produces a therapeutic effect that is greater than the additive effect of the PI3K inhibitor and the second agent.

In the present application, the "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the test compositions disclosed herein is contemplated. Supplementary active ingredients may also be added to the pharmaceutical composition.

In this application, the term "tumor" generally refers to any neoplastic cell growth and proliferation, either malignant or benign, as well as any pre-cancerous and cancerous cells and tissues. In the present application, the term "neoplastic" refers to any form of abnormally regulated or unregulated cell growth, either malignant or benign, resulting in abnormal tissue growth. Thus, "neoplastic cells" include malignant and benign cells with abnormally regulated or unregulated cell growth. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to in this application. In some embodiments, a tumor may refer to a body mass containing a plurality of cancer cells, e.g., cells that exhibit characteristics of any of the cancers described herein. In the present application, the tumor may be a solid tumor or a non-solid tumor (e.g., hematological tumor, lymphoma). The term "solid tumor" generally refers to an abnormal tissue growth or mass, which generally does not contain cysts or areas of liquidity. Solid tumors can be benign (non-cancerous) or malignant (cancerous). In the present application, the tumor may be a tumor in which cells and tissues have abnormal expression of PD-1 or PD-L1. In the present application, the tumor may be a tumor in which cells and tissues have abnormal expression or activity of TGF β or TGF β R. In the present application, the tumor may be a tumor with abnormal expression or activity of PI3K in cells and tissues.

In the present application, the term "resistant" generally refers to when the cancer has a reduced response to treatment, e.g., to the extent that the cancer does not respond to treatment. The cancer may be resistant at the beginning of the treatment or it may develop resistance during the treatment. The cancer subject may have one or more mutations that result in its resistance to treatment, or the subject may make such mutations during treatment. The term "refractory" may refer to a cancer for which treatment (e.g., chemotherapeutic drugs, biologies, and/or radiation therapy) has proven ineffective. Refractory cancer tumors can shrink, but to an extent that the treatment is certainly effective. However, in general, tumors remain the same size as before treatment (stable disease), or grow (progressive disease).

In the present application, the terms "treatment" and "treating" generally refer to a method of achieving a beneficial or desired result, including but not limited to a therapeutic benefit. Therapeutic benefits include, but are not limited to, eradication, suppression, reduction, or amelioration of the underlying disorder being treated. In addition, therapeutic benefit is achieved by eradicating the drug that inhibits, reduces, or ameliorates one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the patient, but the patient may still suffer from the underlying disorder.

In the present application, the terms "prevention" and "preventing" generally refer to a method of achieving a beneficial or desired result, including but not limited to a prophylactic benefit. For prophylactic benefit, a pharmaceutical composition can be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more physiological symptoms of a disease, even if the disease has not yet been diagnosed.

In the present application, the term "subject" or "patient" generally refers to humans (i.e., male or female of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young, middle aged, or elderly)) and/or other primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals, including commercially relevant mammals, such as cows, pigs, horses, sheep, goats, cats, and/or dogs; and/or birds, including commercially relevant birds such as chickens, ducks, geese, quail and/or turkeys.

In the present application, the term "about" or "approximately" generally refers to an acceptable error in the determination of a particular value by one of ordinary skill in the art, which depends in part on the manner in which the value is measured or determined. In certain embodiments, the term "about" or "approximately" generally refers to 1,2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" generally means within a given value or range of 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05%.

In the present application, the terms "comprising" or "including" generally refer to an open form, which should be understood to also contain other substances not mentioned. For example, a pharmaceutical combination comprising a phosphoinositide 3-kinase (PI3K) inhibitor and a second therapeutic agent, wherein the second therapeutic agent is an inhibitor of an immune checkpoint, a TGF β inhibitor, a bifunctional immune checkpoint/TGF β inhibitor or a combination thereof. Wherein "comprising" is to be understood as containing, in addition to the phosphoinositide 3-kinase (PI3K) inhibitor and the second therapeutic agent, other substances.

Detailed Description

In one embodiment, wherein the PI3K inhibitor may comprise: 2- (1- (9H-purin-6-ylamino) propyl) -3- (3-fluorophenyl) -4H-chromen-4-one, or an enantiomer, mixture of enantiomers, mixture of two or more diastereomers, or isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof.

In one embodiment, wherein the PI3K inhibitor may be (S) -2- (1- (9H-purin-6-ylamino) propyl) -3- (3-fluorophenyl) -4H-chromen-4-one (Tenalisib, also known as CN401) or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof.

In one embodiment, the pharmaceutical combination may include the dual PD-L1/TGF β inhibitor and a PI3K inhibitor.

In certain embodiments, wherein the PD-L1/TGF β dual inhibitor may be those described in PCT/CN 20191245635, the entire contents of which are incorporated herein by reference. For example, the PD-L1/TGF β dual inhibitor may be WBP1126 (also referred to as CN202), or a variant or biological analog thereof. The WBP1126 may comprise two polypeptides comprising, in order from N-terminus to C-terminus, the heavy chain variable region (VH), CH2, CH3 domain of an anti-PD-L1 antibody, and TGF β RII or a functionally active fragment thereof, the C-terminus of the CH3 domain being linked to the N-terminus of the TGF β RII or a functionally active fragment thereof by a linker; for example, the polypeptide may have an amino acid sequence shown in table 1:

TABLE 1

In certain embodiments, wherein the PD-L1/TGF β dual inhibitor comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises at least: (i) the heavy chain variable region of an anti-PD-L1 antibody; and (ii) TGF β RII or a functionally active fragment thereof; wherein the second polypeptide comprises at least the light chain variable region of an anti-PD-L1 antibody; wherein the heavy chain variable region of the first polypeptide and the light chain variable region of the second polypeptide, in combination, are capable of specifically binding PD-L1.

In certain embodiments, wherein the PD-L1/TGF β dual inhibitor further comprises a linker linking the C-terminus of the heavy chain variable region of the anti-PD-L1 antibody or antigen-binding fragment thereof to the N-terminus of the TGF β RII or functionally active fragment thereof.

In certain embodiments, wherein the PD-L1/TGF β dual inhibitor comprises a first polypeptide and a second polypeptide, wherein:

the first polypeptide comprises a heavy chain variable region (VH) of an anti-PD-L1 antibody, a CH1 structural domain and TGF beta RII or a functional active fragment thereof from N terminal to C terminal in sequence, wherein the C terminal of the CH1 structural domain is connected with the N terminal of the TGF beta RII or the functional active fragment thereof through a linker; and the second polypeptide comprises, from N-terminus to C-terminus, a light chain variable region (VL) and a light chain constant region (CL) of the anti-PD-L1 antibody.

In certain embodiments, wherein the dual PD-L1/TGF β inhibitor comprises a first polypeptide and a second polypeptide, wherein:

the first polypeptide comprises a heavy chain variable region (VH), a CH1, a CH2, a CH3 domain and TGF beta RII or a functional active fragment thereof of an anti-PD-L1 antibody from the N terminal to the C terminal in sequence, wherein the C terminal of the CH3 domain is connected with the N terminal of the TGF beta RII or the functional active fragment thereof through a linker; and the second polypeptide comprises, from N-terminus to C-terminus, a light chain variable region (VL), a light chain constant region (CL) of the anti-PD-L1 antibody.

In certain embodiments, wherein the linker is a peptide linker.

In certain embodiments, wherein the first polypeptide of the PD-L1/TGF β dual inhibitor may have at least about 80% sequence identity to a polypeptide of the following molecule having the same function: m7824 or SHR-1701.

In certain embodiments, wherein the second polypeptide of the PD-L1/TGF β dual inhibitor may have at least about 80% sequence identity to a polypeptide of the following molecule having the same function: m7824 or SHR-1701.

In certain embodiments, wherein the PD-L1/TGF β dual inhibitor may be those described in PCT/EP2015052781, PCT/CN2018086451, the entire contents of all of which are incorporated herein by reference.

In certain embodiments, wherein the PD-L1/TGF β dual inhibitor may be selected from M7824, SHR-1701, or variants or biological analogs thereof, or combinations thereof.

In certain embodiments, the PD-L1/TGF β dual inhibitor and the PI3K inhibitor may not be intermixed in the pharmaceutical combination.

In certain embodiments, the PD-L1/TGF β dual inhibitor and the PI3K inhibitor may each be present independently in separate containers.

Pharmaceutical composition and use

In another aspect, the present application provides a pharmaceutical composition comprising the aforementioned PI3K inhibitor and a second therapeutic agent, and optionally one or more pharmaceutically acceptable carriers or excipients.

In certain embodiments, wherein the second therapeutic agent is the aforementioned dual PD-L1/TGF β inhibitor.

In certain embodiments, wherein the PI3K inhibitor is the aforementioned PI3K delta/gamma dual inhibitor.

In certain embodiments the pharmaceutical composition comprises a dual PD-L1/TGF β inhibitor and a dual PI3K δ/γ inhibitor.

For example, the pharmaceutical composition may include: i) WBP1126 or a variant or biological analog thereof; and

ii) Tenalisib or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof.

In certain embodiments, wherein the second therapeutic agent is an anti-PD-L1 antibody as described previously and the PI3K inhibitor is a PI3K delta/gamma dual inhibitor as described previously.

For example, the pharmaceutical composition may include: i) nivolumab, pembrolizumab, pidilizumab, or a combination thereof; and ii) Tenalisib or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof.

In certain embodiments, wherein the second therapeutic agent is the aforementioned dual PD-L1/TGF β inhibitor, the PI3K inhibitor is a PI3K α inhibitor.

For example, the pharmaceutical composition may comprise: i) WBP1126 or a variant or biological analog thereof; and

ii) Alplisib or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof.

In certain embodiments, wherein the second therapeutic agent and the PI3K inhibitor are present in a single or separate dosage form.

In certain embodiments, wherein the pharmaceutical composition comprises, per unit dosage form (unit dose form), a dose of 1 to 1000mg of a first therapeutic agent, e.g., a PI3K inhibitor active (e.g., CN401(Tenalisib)), e.g., 1,2, 3,4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000mg or a value between any two of the foregoing values; and, a dosage of 1-1000mg of a second therapeutic agent, e.g., any one of a PD-L1 inhibitor, a TGF β inhibitor, a PD-L1 inhibitor, a PD-L1/TGF β dual inhibitor, e.g., CN202(WBP1126), or a PD-1/TGF β dual inhibitor, or a combination thereof, per unit dosage form (unit dosage form), e.g., a value between any two of 1,2, 3,4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000mg, or more.

In certain embodiments, the pharmaceutical composition may further comprise a third active agent.

In certain embodiments, wherein the third active agent may be selected from any one of vincristine, vinblastine, vindesine, etoposide, docetaxel, paclitaxel (e.g., albumin bound paclitaxel), irinotecan, vinorelbine, mitoxantrone, vinflunine, topotecan, or any combination thereof.

In another aspect, the present application provides a method of treating and/or preventing a tumor comprising administering to a subject in need thereof: an effective amount of a pharmaceutical combination of the foregoing, or a pharmaceutical composition of the foregoing.

In certain embodiments, the method comprises administering to a subject in need thereof: an effective amount of the aforementioned PI3K inhibitor and the aforementioned inhibitor of an immune checkpoint. In certain embodiments, the method comprises administering to a subject in need thereof: an effective amount of the aforementioned PI3K inhibitor and the aforementioned TGF β inhibitor. In certain embodiments, the method comprises administering to a subject in need thereof: an effective amount of the aforementioned PI3K inhibitor, the aforementioned TGF β inhibitor, and the aforementioned inhibitor of an immune checkpoint.

For example, the method may comprise administering to a subject in need thereof: an effective amount of the foregoing PI3K inhibitor, e.g., the foregoing (S) -2- (1- (9H-purin-6-ylamino) propyl) -3- (3-fluorophenyl) -4H-chromen-4-one, or a pharmaceutically acceptable salt thereof, and the foregoing anti-PD-L1 antibody, e.g., Atezolizumab, Avelumab, BMS-936559, MPDL3280A, durvalumab, or a combination thereof.

For example, the method may comprise administering to a subject in need thereof: an effective amount of an aforementioned PI3K inhibitor, e.g., an aforementioned (S) -2- (1- (9H-purin-6-ylamino) propyl) -3- (3-fluorophenyl) -4H-chromen-4-one, or a pharmaceutically acceptable salt thereof, and an aforementioned TGF β inhibitor, e.g., Fressolimumab, SB525334, or a combination thereof.

For example, the method may comprise administering to a subject in need thereof: an effective amount of the foregoing PI3K inhibitor, e.g., the foregoing (S) -2- (1- (9H-purin-6-ylamino) propyl) -3- (3-fluorophenyl) -4H-chromen-4-one, or a pharmaceutically acceptable salt thereof; the foregoing TGF β inhibitors, e.g., Fresolimumab, SB525334, or combinations thereof; the aforementioned anti-PD-L1 antibodies, for example, Atezolizumab, Avelumab, BMS-936559, MPDL3280A, durvalumab, or combinations thereof.

For example, the method may comprise administering to a subject in need thereof: an effective amount of a PI3K inhibitor as hereinbefore described, for example (S) -2- (1- (9H-purin-6-ylamino) propyl) -3- (3-fluorophenyl) -4H-chromen-4-one as hereinbefore described, or a pharmaceutically acceptable salt thereof; the foregoing TGF β inhibitors, e.g., Fresolimumab, SB525334, or combinations thereof; the aforementioned anti-PD-1 antibodies, e.g., nivolumab, pembrolizumab, pidilizumab, or a combination thereof.

In certain embodiments, the method comprises administering to a subject in need thereof: an effective amount of the aforementioned PI3K inhibitor and the aforementioned PD-L1/TGF β dual inhibitor.

For example, the method may comprise administering to a subject in need thereof: an effective amount of a PI3K inhibitor as described previously, e.g., (S) -2- (1- (9H-purin-6-ylamino) propyl) -3- (3-fluorophenyl) -4H-chromen-4-one as described previously; and the aforementioned dual PD-L1/TGF β inhibitors, such as WBP1126, M7824, SHR-1701, or variants or biological analogs thereof, or combinations thereof.

In certain embodiments, wherein the PI3K inhibitor may be administered concurrently with the dual PD-L1/TGF β inhibitor.

In certain embodiments, wherein the PI3K inhibitor may be administered after the PD-L1/TGF β dual inhibitor.

In certain embodiments, wherein the PI3K inhibitor may be administered prior to the PD-L1/TGF β dual inhibitor.

In certain embodiments, further comprising administering to a subject in need thereof an effective amount of the aforementioned small molecule TGF β inhibitor.

In certain embodiments, wherein the tumor may comprise a solid tumor and a non-solid tumor.

In certain embodiments, wherein the tumor may comprise a tumor with aberrant PI3K expression.

In certain embodiments, wherein the tumor may comprise a tumor with aberrant PD-L1 expression.

In certain embodiments, wherein the tumor may comprise a tumor with aberrant TGF β expression.

In certain embodiments, the tumor may comprise a tumor of the digestive tract, melanoma, or lymphoma.

In some embodiments, the methods of the present application delay resistance compared to the time resistance typically develops when either the agent or the inhibitor is used alone as a monotherapy treatment for the subject. In some embodiments, resistance is delayed for at least 2 weeks, e.g., at least 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, 12 months, 1 year, 2 years, 4 years, 6 years, 8 years, or longer. In some embodiments, according to the method, the remission (e.g., complete remission or partial remission) is extended as compared to the time that remission generally persists when the subject is treated with either the agent or the inhibitor alone as monotherapy. In some embodiments, remission (e.g., complete remission or partial remission) is extended for at least 2 weeks, e.g., at least 2 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, 12 months, 1 year, 2 years, 4 years, 6 years, 8 years, or more.

In some embodiments, the addition of a PI3K inhibitor or a second agent to a treatment regimen increases or restores sensitivity to an agent that is resistant to cancer. For example, in some embodiments, the addition of a second agent to a treatment regimen increases or restores sensitivity to a cancer-resistant PI3K inhibitor.

In certain embodiments, wherein remission of the cancer in the subject is prolonged.

In certain embodiments, wherein the subject experiences complete remission of the cancer.

In some embodiments, the methods described herein comprise selecting a subject for treatment with a combination of a PI3K inhibitor and a second agent. In certain embodiments, a subject (e.g., a patient having a cancer, such as a cancer described herein) is selected for treatment with the combination based on the MRD in the subject. In certain embodiments, the selection is above a preselected level based on the presence of MRD (e.g., 1 malignant cell out of 100 normal cells, 1 malignant cell out of 1000 normal cells, or 1 malignant cell out of 10,000 normal cells). Methods for monitoring minimal residual disease negative (MRD) are known in the art. See, e.g., Zhou, J, et al, Blood,2007,110: 1607-. Such methods include DNA-based tests or RNA-based tests. In certain embodiments, MRD is monitored using flow cytometry, sequencing, or PCR.

In another aspect, the present application provides a pharmaceutical combination as described above or a pharmaceutical composition as described above for use in the treatment and/or prevention of a tumor.

In certain embodiments, the method comprises contacting the tumor with an inhibitor of PI3K as described above and an inhibitor of an immune checkpoint as described above. In certain embodiments, the method comprises contacting the tumor with the aforementioned PI3K inhibitor and the aforementioned TGF β inhibitor. In certain embodiments, the method comprises contacting the tumor with the aforementioned PI3K inhibitor, the aforementioned inhibitor of an immune checkpoint, and the aforementioned TGF β inhibitor.

In another aspect, the present application provides a method of inhibiting tumor growth, the method comprising contacting a tumor with the aforementioned PI3K inhibitor and the aforementioned dual PD-L1/TGF β inhibitor.

In another aspect, the present application provides a kit comprising: (1) a first container, and the aforementioned PI3K inhibitor located in the first container; (2) a second container, and the aforementioned dual inhibitor of PD-L1/TGF β located in the second container.

In another aspect, the present application provides a kit, which may include: (1) a first container, and the aforementioned PI3K inhibitor located in the first container; (2) a second container, and an inhibitor of the aforementioned immune checkpoint or an inhibitor of the aforementioned TGF β located in said second container.

In certain embodiments, the kit further comprises (3) a third container, and an inhibitor of the aforementioned immune checkpoint or the aforementioned TGF β inhibitor located in the third container, the agent in the third container being different from the agent in the second container.

In certain embodiments, the dosage form of the drug in the kit can be an oral dosage form or an injectable dosage form.

In certain embodiments, the kit can further comprise instructions.

Without wishing to be bound by any theory, the following examples are only intended to illustrate the pharmaceutical compositions, methods of preparation, uses, etc. of the present application and are not intended to limit the scope of the invention of the present application.

Examples

Materials and methods

The animal experiment is carried out according to the AAALAC requirement, and is approved by an IACUC center of the Bikea animal experiment to carry out the animal experiment. The method comprises purchasing 6-8 weeks old Balb/c mice (16-20 g) from Beijing Wittingle laboratory animal technology, Inc., subcutaneously inoculating 50 ten thousand A20 cells into each mouse, and inoculatingIs planted on the right rear side of the back of the mouse. When the tumor volume grows to about 60mm ³ The left and right groups were randomly assigned to the control group and the experimental group.

Mice were euthanized when morbidity or weight loss was observed to be greater than or equal to 20%. Tumors were measured twice weekly with calipers until final sacrifice. When the tumor size reaches about 2000mm ³ Or when there was an animal health problem (20% ulcer of the tumor area), the animal would be euthanized and the death date recorded. Solid tumor volume was estimated from two-dimensional tumor measurements and calculated according to the following equation:

TV, Tumor volume. TV (mm) ³ ) Length (mm) x width ² (mm ² )]/2

The median percent regression (regression) for a given group of heliostats was then obtained by taking the median of the individual percent regressions calculated for each animal in the group on that day. The date of calculation was determined on the day of calculation of Δ T/Δ C (i.e., the median proportion of change in tumor volume from baseline between experimental and experimental groups), unless the median percent regression does not represent activity for that group. In that case, the first day when the median percentage subsided the greatest was determined as the day. Regression was defined as Partial (PR) if the tumor volume decreased to 50% of the tumor volume at the start of treatment. When the tumor volume is less than 14mm ³ Or when it was not recordable, Complete Regression (CR) was considered to have been achieved.

RTV Relative Tumor volume. RTV-V _t /V ₀ 。V _t : tumor volume at the end of one experimental period (treatment group). V ₀ : tumor volume at the beginning of the experiment

TGI: tgi [1-RTV (experimental)/RTV (control) ]) 100%

Statistical analysis

Changes in tumor volume compared to baseline were subjected to two-way ANOVA patterns with factor treatment and days (replicates). In the case of significant treatment-by-day interactions or treatment effects, a comparative analysis with Bonferroni-Holm correction multiplicity was then performed to compare all experimental groups with the control group on each of days 8 to 27. Changes in tumor volume from baseline were calculated for each animal and on each day by subtracting the tumor volume on the day of first treatment from the tumor volume on the indicated observation day. Because of the heterogeneity of differences observed between groups, a Composite Symmetric (CS) covariance structure with group-options was chosen for the ANOVA type model (SAS Institute Inc. (2008) SAS/STAT 9.2 user guide, Cary NC). All statistical analyses were performed using SAS version v9.2 software. A probability of less than 5% (p <0.05) was considered significant.

Example 1 combination study of PI3K inhibitor with different doses of TGF β/PDL1 bispecific antibody

Example 1.1 combination study of CN401(Tenalisib) and CN202(WBP1126) (5mg/kg) in the A20 mouse B cell lymphoma model (Experimental design, see Table 2)

6-8 week old balb/c mice (18-20g) were purchased from Beijing Wintolite laboratory animal technology, Inc., and each mouse was subcutaneously inoculated with 50 ten thousand A20 cells and inoculated on the right rear side of the back of the mouse. When the tumor volume grows to about 62mm ³ The treatment is started. Wherein CN401 is orally administered at 150mg/kg twice daily for 3 weeks.

CN202 was administered by intraperitoneal injection, 3 times a week at a dose of 5mg/kg for 3 weeks. The control group received vehicle without active product (0.5% MC and PBS). Tumor size and body weight were measured three times per week. The animal experiment is carried out according to the AAALAC requirement, and is approved by an IACUC center of the Bikea animal experiment to carry out the animal experiment.

TABLE 2

Group of	Number of	Administration mode
			1	8	Blank control group (PBS i.p.tiw.times.3 weeks + 0.5% MC p.o.bid.times.3 weeks)
2	8	CN401 150mg/kg,p.o.bid×3weeks
			3	8	CN202 5mg/kg,i.p.tiw×3weeks
4	8	CN401 150mg/kg,p.o.bid×3weeks+CN202 5mg/kg,i.p.tiw×3weeks

The results of the tumor growth curve and the body weight growth curve are shown in FIGS. 1A-1F; TGI, p-value and number of fully responsive mice in the treatment group compared to the blank group on day 22 are shown in table 3.

As a result: in this study, CN401 in combination with CN202 had a synergistic effect on tumor inhibition in a20 mouse B cell lymphoma model (p 0.0001, TGI 89%, CR 4/8), and the mice showed good tolerance to the dosing regimen.

TABLE 3

Example 1.2 combination of CN401 and CN202(15mg/kg) in the A20 mouse B-cell lymphoma model (see Table 4 for experimental design)

The test method of example 1.1 was referenced, except that the CN202 dosage was replaced from 5mg/kg to 15 mg/kg.

TABLE 4

Group of	Number of	Mode of administration
			1	8	Blank control group (PBS i.p.tiw.times.3 weeks + 0.5% MC p.o.bid.times.3 weeks)
2	8	CN401 150mg/kg,p.o.bid×3weeks
			3	8	CN202 15mg/kg,i.p.tiw×3weeks
4	8	CN401 150mg/kg,p.o.bid×3weeks+CN202 15mg/kg,i.p.tiw×3weeks

The results of the tumor growth curve and the body weight growth curve are shown in FIGS. 2A-2F; TGI, p-values and number of fully responsive mice in the treatment group compared to the blank group on day 22 are shown in table 5.

As a result: in this study, CN401 in combination with CN202 had a synergistic effect on tumor suppression in a20 mouse B-cell lymphoma model (p 0.023, TGI 71%, CR 5/8), and the mice showed good tolerance to the dosing regimen.

TABLE 5

Example 2 combination study of TGF-beta/PDL 1 bispecific antibodies with different forms of PI3K inhibition

Example 2.1 combination study of CN202(WBP1126) and CN401(Tenalisib) (see Table 6 for design of test)

6-8 week old balb/c mice (18-20g) were purchased from Peking Wintonlifa laboratory animal technology Ltd, and each mouse was subcutaneously inoculated with 50 ten thousand A20 cells and inoculated on the right back side of the back of the mouse. When the tumor volume grows to about 80mm ³ The treatment is started. Wherein CN202 was administered by intraperitoneal injection at a dose of 5mg/kg 3 times a week for 3 weeks. CN401 was orally administered at 150mg/kg twice daily for 3 weeks. The control group received vehicle without active product (0.5% MC and PBS). Tumor size and body weight were measured three times per week. The animal experiment is carried out according to the AAALAC requirement, and is approved by an IACUC center of the Bikea animal experiment to carry out the animal experiment.

TABLE 6

Group of	Number of	Administration mode
			1	7	Blank control group (PBS i.p.tiw.times.3 weeks + 0.5% MC p.o.bid.3 weeks)
2	7	CN202 5mg/kg i.p.tiw×3weeks
			3	7	CN401 150mg/kg p.o.bid×3weeks
4	7	CN202 5mg/kg i.p.tiw×3weeks+CN401 150mg/kg p.o.bid×3weeks

The results of the tumor growth curve and the body weight growth curve are shown in fig. 4A-4F; TGI, p-values and number of fully responsive mice in the treatment group compared to the blank group on day 21 are shown in table 7.

As a result: in this study, CN202 in combination with CN401 had a synergistic effect on tumor suppression in a20 mouse B-cell lymphoma model (p ═ 0.0009, TGI ═ 85%, CR ═ 6/7), and the mice showed good tolerance to the dosing regimen.

TABLE 7

Example 2.2 combination study of CN202(WBP1126) with Alpelisib (design of test see Table 8)

Reference is made to the test method of example 2.1, except that the PI3K inhibitor is administered orally by CN401(Tenalisib) twice daily at 150mg/kg for 3weeks instead of Alpelisib being administered orally once daily at 50mg/kg for 3 weeks.

TABLE 8

Group of	Number of	Administration plan
			1	7	Blank control group (PBS i.p.tiw.times.3 weeks + 0.5% MC p.o.bid.3 weeks)
2	7	CN202 5mg/kg i.p.tiw×3weeks
			3	7	Alpelisib 50mg/kg p.o.qd×3weeks
4	7	CN202 5mg/kg i.p.tiw×3weeks+Alpelisib 50mg/kg p.o.qd×3weeks

The results of the tumor growth curve and the body weight growth curve are shown in FIGS. 5A-5F; TGI, p-values and number of fully responsive mice in the treatment group compared to the blank group on day 21 are shown in table 9.

As a result: in this study, CN202 in combination with Alpelisib showed a synergistic effect on tumor suppression in the a20 mouse B-cell lymphoma model (p 0.0002, TGI 85% and CR 3/7), and the mice also showed good tolerance to the dosing regimen.

TABLE 9

Example 3 Co-administration of CN401 with anti-PD-L1 antibody in A20 mouse B-cell lymphoma model (for experimental design, see Table 10)

6-8 week old balb/c mice (18-20g) were purchased from Peking Wintonlifa laboratory animal technology Ltd, and each mouse was subcutaneously inoculated with 50 ten thousand A20 cells and inoculated on the right back side of the back of the mouse. When the tumor volume grows to about 62mm ³ The treatment is started. CN401 was orally administered at 150mg/kg twice daily for 3 weeks. Wherein the anti-PD-L1 antibody (Atezolizumab) was administered by intraperitoneal injection at a dose of 5mg/kg 2 times per week for 3 weeks. The control group received vehicle without active product (0.5% MC and PBS). Tumor size and body weight were measured three times per week. The animal experiment is carried out according to the AAALAC requirement and is approved by the animal experiment center IACUC of Bikean.

Watch 10

The results of the tumor growth curve and the body weight growth curve are shown in FIGS. 6A-6F; TGI, p-values and number of fully responsive mice in the treatment group compared to the blank group on day 22 are shown in table 11.

As a result: in this study, CN401 in combination with an anti-PD-L1 antibody had a synergistic effect on tumor inhibition in a20 mouse B cell lymphoma model (p 0.026, TGI 63%, CR 3/8), and the mice showed good tolerance to the dosing regimen.

TABLE 11

Example 4 study of combination of CN401, CN202 and Albumin paclitaxel (nab-pac) in EMT-6 tumor model (design of experiment, see Table 12)

6-8 week old balb/c mice (18-20g) were purchased from Peking Wintonlihua laboratory animal technology, Inc., and each mouse was inoculated subcutaneously (supplementary test procedure). The control group received vehicle without active product (0.5% MC and PBS). Tumor size and body weight were measured three times per week. The animal experiment is carried out according to the AAALAC requirement, and is approved by an IACUC center of the Bikea animal experiment to carry out the animal experiment.

TABLE 12

The results of the tumor growth curve and the body weight growth curve are shown in FIG. 7.

As a result: in this study, CN401 in combination with CN202, albumin paclitaxel, had a synergistic effect on tumor inhibition in the EMT-6 tumor model (CR-4/8).

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Claims

1. A pharmaceutical combination comprising a phosphoinositide 3-kinase (PI3K) inhibitor and a second therapeutic agent, wherein the second therapeutic agent is a bifunctional immune checkpoint/TGF β inhibitor or a combination thereof; preferably, said bifunctional immune checkpoint/TGF β inhibitor is selected from the group consisting of a combination of a PD-L1/TGF β dual inhibitor and a PI3K inhibitor; or a combination of a dual PD-1/TGF β inhibitor and a PI3K inhibitor.

2. The pharmaceutical combination according to claim 1, wherein the dual PD-L1/TGF β inhibitor is a fusion protein.

3. The pharmaceutical combination of claim 1 or 2, wherein the PD-L1// TGF β dual inhibitor comprises a PD-L1 targeting moiety and a TGF β receptor domain comprising transforming growth factor beta receptor II (TGF β RII) or a functionally active fragment thereof.

4. The pharmaceutical combination according to any one of claims 1-3, wherein the PD-L1// TGF β dual inhibitor comprises a polypeptide, wherein the polypeptide comprises at least: (i) the heavy chain variable region of an anti-PD-L1 antibody; and (ii) TGF β RII or a functionally active fragment thereof.

5. The pharmaceutical combination of any one of claims 1-4, wherein the anti-PD-L1 antibody heavy chain variable region comprises HCDR1, HCDR2, HCDR 3; wherein the HCDR1 comprises an amino acid sequence having at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO. 1, the HCDR2 comprises an amino acid sequence having at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO. 2, and the HCDR3 comprises an amino acid sequence having at least about 70% sequence identity to the amino acid sequence set forth in SEQ ID NO. 3; or

6. The pharmaceutical combination of any one of claims 1-5, wherein the anti-PD-L1 antibody heavy chain variable region comprises HCDR1, HCDR2, HCDR3, said HCDR1 comprising the amino acid sequence set forth in SEQ ID No. 1 or an amino acid sequence obtained by amino acid addition, deletion or substitution reactions having NO more than 2 amino acid differences from the amino acid sequence set forth in SEQ ID No. 1; the HCDR2 comprises an amino acid sequence shown in SEQ ID NO. 2 or an amino acid sequence which is obtained by amino acid addition, elimination or substitution reaction and has NO more than 2 amino acid differences with the amino acid sequence shown in SEQ ID NO. 2; the HCDR3 comprises an amino acid sequence shown in SEQ ID NO. 3 or an amino acid sequence which is obtained by amino acid addition, elimination or substitution reaction and has NO more than 2 amino acid differences with the amino acid sequence shown in SEQ ID NO. 3.

7. The pharmaceutical combination of any one of claims 1-6, wherein the anti-PD-L1 antibody heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: (a) 4, an amino acid sequence shown as SEQ ID NO; (b) an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 4; (c) an amino acid sequence having 1 or more differences from the amino acid sequence shown in SEQ ID NO. 4, which is obtained by addition, elimination or substitution reaction; and (d) an amino acid sequence having at least about 80% sequence identity to the heavy chain variable region of: atezolizumab, Avelumab, BMS-936559, MPDL3280A or durvalumab.

8. The pharmaceutical combination according to any one of claims 1-7, wherein the TGF β RII or functionally active fragment thereof comprises:

(a) an amino acid sequence shown as SEQ ID NO. 6;

9. The pharmaceutical combination of any one of claims 1-8, wherein the polypeptide further comprises a linker linking the C-terminus of the heavy chain variable region of the anti-PD-L1 antibody or antigen-binding fragment thereof to the N-terminus of the TGF β RII or functionally active fragment thereof.

10. The pharmaceutical combination of any one of claims 1-9, the polypeptide further comprising a CH2, a CH3 domain, the polypeptide comprising, in order from N-terminus to C-terminus, the heavy chain variable region (VH), the CH2, the CH3 domain, and the TGF β RII of the anti-PD-L1 antibody, or a functionally active fragment thereof, the C-terminus of the CH3 domain being linked to the N-terminus of the TGF β RII or a functionally active fragment thereof by a linker; preferably, the CH2, CH3 domains are derived from IgG.

11. The pharmaceutical combination of claim 9, wherein the linker is a peptide linker; preferably, the amino acid sequence of the peptide linker is (G) ₄ S) _x Wherein x is any integer from 3 to 6; more preferably, the peptide linker comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence shown as SEQ ID NO. 5; (b) amino acids having at least about 85%, 90%, 95% or 99% sequence identity to the amino acid sequence shown in SEQ ID NO. 5A sequence; and (c) an amino acid sequence having 1 or more difference from the amino acid sequence shown in SEQ ID NO. 5, which is obtained by addition, elimination or substitution reaction.

12. The pharmaceutical combination of claims 1-11, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of seq id no: (a) the amino acid sequence shown as SEQ ID NO. 7; (b) an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 7; (c) an amino acid sequence having 1 or more differences from the amino acid sequence shown in SEQ ID NO. 7, which is obtained by addition, deletion or substitution reaction.

13. The pharmaceutical combination of any one of claims 1-12, wherein the PI3K inhibitor is a PI3K delta/gamma dual inhibitor; preferably, the PI3K inhibitor comprises 2- (1- (9H-purin-6-ylamino) propyl) -3- (3-fluorophenyl) -4H-chromen-4-one, or an enantiomer, a mixture of enantiomers, a mixture of two or more diastereomers, or an isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof; more preferably, the PI3K inhibitor comprises (S) -2- (1- (9H-purin-6-ylamino) propyl) -3- (3-fluorophenyl) -4H-chromen-4-one, or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof.

14. The pharmaceutical combination according to any one of claims 1-13, wherein the second therapeutic agent and the PI3K inhibitor are not mixed with each other in the pharmaceutical combination or are separately present in separate containers.